1. Field of the Invention
The present invention relates to novel galactosyl maltooligosaccharide derivatives with excellent steerage stability which can be used as a substrate for measurement of .alpha.-amylase activity. The present invention further relates to a method for preparation of the above derivatives and a method for measurement of .alpha.-amylase activity using the derivatives.
2. Description of the Related Art
It is quite important for clinical diagnosis to measure the activity of .alpha.-amylase contained in body fluids of humans such as saliva, pancreatic carcinoma or parotitis, especially when these levels are remarkably increased. Therefore, the .alpha.-amylase activity is an important indicator for clinical diagnosis.
Among of various substrates for assay methods of .alpha.-amylase activity reported previously, maltooligosaccharides and their derivatives having definite chemical structures are widely used these days.
In most of these methods, enzyme activity is determined by measuring glucose or adsorption value of various aglycons (e.g., p-nitrophenol), which are liberated from maltooligosaccharides or their derivatives by the simultaneous action of .alpha.-amylase and coupled enzymes (.alpha.-and/or .beta.-glucosidases) during the reaction, respectively.
However, it is very difficult to keep the initial quality of these substrates at a constant level, during preservation when the mixture of the coupled enzyme and substrate is stored as a solution, because these coupled enzymes have the ability to hydrolyze the substrates even if these preparation are sufficiently free from .alpha.-amylase activity. In order to improve the conventional methods, it has been tried to use some substrates, which are obtained by chemical modification of the nonreducing end of glucose of the maltooligosaccharides or their derivatives, to prevent hydrolysis by these coupled enzymes during preservations.
These kinds of chemically modified substrates are not hydrolyzed by coupled enzymes such as .alpha.-D-glucosidase and glucoamylase.
Accordingly, it is possible to obtain a stable preparation containing substrate and the coupled enzyme for the assay of .alpha.-amylase activity. These substrates prevent the significant increase of blank value even if they are preserved for a long time.
For example, methods using nonreducing end substituted maltooligosaccharides with a carboxymethyl group and 2-pyridylamino residue as a substrate are known (see Japanese Patent Disclosure No.59-31699 (JP-A-31699/1984), No. 59-51800(JP-A-51800/1984) and 61-83195 (JP-A-83195/1986)). However, complicated steps are required for the preparation of these kinds of substrates. For instance, after random chemical modification of glucose molecules in dextrin or amylose, the partially modified macromolecular saccharides are hydrolyzed with bacterial saccharifying type .alpha.-amylase and glucoamylase to obtain nonreducing end substituted maltooligosaccharides, and the resulting maltooligosaccharides are further fractionated in a certain chromatographic fractionated manner with certain kinds of resins to prepare modified maltooligosaccharides having definite a degree of polymerization. Then, these nonreducing end substituted maltooligosaccharides are transferred to certain acceptors such as p-nitrophenyl .alpha.-D-glucoside with cyclomaltodextrin glucanotransferase to obtain maltooligosaccharides of which both terminal glucose molecules are modified. And, the resulting product both reducing-and nonreducing-end substituted maltooligosaccharides are fractionated further by chromatographic methods to obtain the modified saccharides having a definite chemical structure and molecular mass. Due to these complicated procedures for preparation, the yield of the desired derivatives is low and therefore, this method is impractical.
There have been many patents regarding the assay method for .alpha.-amylase activity using maltooligosaccharide derivatives of which the nonreducing end glucose molecule was chemically modified, and the opposite end glucose was linked to some kinds of chromophore (see Japanese Patent Disclosure No.60-54395 (JP-A-54935/1985), No.60-87297(JP-A-87297/1985), No.60-237998(JP-A-237998/1985), No.61-3299(JP-A-63299/1986), No.63-301892(JP-A-301892/1988) and No.1-157996(JP-A-157996/1989)).
In these methods, a substituent in such as benzylidene, ethylidene, iso-propylidene, halogen, alkyl, phenyl, benzyl or ketobutyl is introduced into the nonreducing end glucose of a maltooligosaccharide and the products are used as substrates. However, the chemical synthesis methods in which the nonreducing end is specifically modified require complicated steps, for example, an acetylation and a deacetylation step is required and therefore the yield of the final products is low.
As mentioned above, preparation of maltooligosaccharide derivatives with the modified nonreducing end glucose is difficult, and also it is not easy to use them practically as a substrate for the assay of .alpha.-amylase activity.
An object of the invention is to provide novel maltooligosaccharide derivatives which are easy to prepare and stable against the coupled enzymes when they are used as a substrate for the assay method of .alpha.-amylases activity.
A further object of the invention is to provide a process for preparation to the above novel maltooligosaccharide derivatives and a method for measurement of the activity of .alpha.-amylase.